1. Field of the Invention
The present invention relates to chemiluminescent compounds. More particularly, the present invention concerns stable, triggerable chemiluminescent 1,2-dioxetanes. Even more particularly, the present invention concerns new chemiluminescent 1,2-dioxetane compounds derived from the oxidation of novel alkenes prepared by the coupling of substituted aromatic esters or ketones and spiro-fused ketones with or without a π-electron system or a carbon-carbon double bond in the ring.
2. Prior Art
Chemiluminescent compounds, their preparation and their uses have been long documented in the prior art. These “high energy” molecules store sufficient energy to generate, or fragmentation, electronically excited carbonyl products which are responsible for the observed chemiluminescence. Dioxetanes and especially, 1,2-dioxitanes and eminently useful to detect the presence, as well as the absence, of certain enzymes in fluids such as blood and the like because of their chemiluminescence. Thus, 1,2 dioxetanes are eminently useful in doing medical assays.
Generally, 1,2-dioxetanes are thermally labile substances having a wide range of stability which decompose on heating and emit light, and correspond to the following formula (1):

Where each R corresponds to any one of a multitude of organic moieties widely reported in the prior art, as detailed herebelow. As noted these 1,2-dioxetanes have a wide range of stability. For example, the prior art, as found in: (a) K. W. Lee, L. A. Singer and K. D. Legg, J. Org. Chem., 41, 2685(1976); (b) F. McCapra, I. Beheshti, A. Burford, R. A. Hanu and K. A. Zaklika, J. Chem. Soc., Chem. Commun., 944(1977); and (c) J. H. Wieringa, J. Strating, H. Wynberg and W. Adam, Tet. Lett., 169 (1972); respectively, disclose the following 1,2-dioxetanes of different stability:

Although these high energy compounds are all spiro-substituted 1,2-dioxetanes, spiroadamantane substitution exerts a tremendous stabilizing effect on these four-membered ring peroxides. The lower activation energy (EA) of the dioxetanes of formulae (3) and (4) above is explained by the donation of charge from nitrogen to the dioxetane ring. The dioxetane of formula (5) above decomposes at 150° C. and has a half life at 25° C. more than 20 years.
A 1,2-dioxetane (6) below, dispiro[adamantane2,3-[1,2-] dioxetane-4,9-fluorene] was isolated as crystals and described by W. Adam and L. A. A. Encarnacion, Chem. Ber., 115, 2592 (1982).

The stability of 1,2-dioxetanes (5) and (6) was described on the basis of bulky and rigid spiro nature of the adamantane group.
The first stable and enzymatic triggerable 1,2-dioxetane was synthesised by the oxidation of (6-acetoxy-2-naphthyl) methoxy methyleneadamantane as reported by A. P Schaap, R. S. Handley and B. P Giri, Tet. lett., 935 (1987). This 1,2-dioxetane utlizes aryl esterase emzyme to catalyze the cleavage of the acetate group of a naphthylacetate-substituted-1,2-dioxetane and produce chemiluminescence in aqueous buffers at ambient temperature by the following sequence:

Several other stabilized 1,2-dioxetanes and their use as enzyme substrates have been disclosed in the literature. See, inter alia, A. P. Schaap, T. S. Chen, R. S. Handley, R. DeSilva and B. R Giri, Tet. Lett., 1155(1987); A. P. Schaap, M. D. Sandison and R. S. Handley, Tet. Lett., 1159 (1987); U.S. Pat. No. 4,962,192; U.S. Pat. No. 4,978,614; U.S. Pat. No. 5,386,017; U.S. Pat. No. 5,721,370, the disclosures of which are hereby incorporated by reference.
These several other 1,2-dioxetanes, generally, have the following general structures:
wherein
is a non-active site and which is selected from the group of polycyclic alkyl groups containing 6 to 30 carbon atoms, OX is an oxy group substituted on an aryl ring which forms an unstable oxide intermediate 1,2-dioxetane compound when triggered to remove X by an activating agent selected from the group consisting of an acid, a base, a salt, an enzyme and an inorganic or organic catalyst, and electron donor source, and X is a chemically labile group which is removed by the activating agents to form light and carbonyl containing compounds, R1 is a lower alkyl containing 1 to 8 carbon atoms, or mixtures thereof, or

where T is a non-active site which is a cycloalkyl or a polycycloalkyl group bonded to the 4-membered ring portion of the dioxetane by a spiro linkage; Y is a fluorescent chromophore; X is a hydrogen, alkyl, aryl arylkyl, alkaryl, heteroalkyl. heteroaryl, cycloalkyl, cycloheteroalkyl, or enzyme cleavable group; and Z is hydrogen or an enzyme cleavable group, provided that at least one of X or Z must be an enzyme cleavable group.
The enzyme cleavable 1,2-dioxetanes of formulae (14), (15) and (16) shown below have been commercialized and used in immuno assays, southern blotting, northern blotting, western blotting, plaque/colony lifts and DNA sequencing.
The 1,2-dioxetane of formula (15) with the chloro substitution in the adamantane ring demonstrates better results in DNA sequencing when compared to dioxetane (14). Dioxetane (16) is more soluble in an aqueous system than 1,2-dioxetane (14) and (15) when a CH3 group is replaced with a CH2CH2CH2COOH.
Other relevant prior art can be found in U.S. Pat. Nos. 5,386,017; 4,962,192; 5,018,827; 5,578,253; 5,004,565; 5,068,339, the disclosures of which are hereby incorporated by reference.
While these prior art compounds provide enzyme cleavable 1,2-dioxetanes, it has been observed that in an aqueous buffer, the luminescence of these molecules is particularity poor, especially when trace amounts of biological materials are sought to be detected. Thus more powerful dioxetanes are needed i.e. dioxetanes having higher levels of chemiluminescence in an aqueous buffer.